Tools to monitor immune cells interactions and their consequences in vitro and in vivo are needed to accelerate development of successful immunotherapies for cancer, HIV disease, and autoimmune disorders. Stable fluorescent labels for cell proteins (CFSE) or membranes (PKH dyes) are such tools. However, high levels of autofluorescence and spectral overlap in the visible region limit their use in vitro, and real time optical imaging in intact animals is precluded by high levels of tissue absorption and scattering. We hypothesize that stable membrane labels that fluoresce in the far red (FR) or near infrared (NIR) will: a) enable more sensitive cell tracking using flow cytometry and confocal microscopy, b) provide novel non-radioactive contrast agents for optical imaging of immune cell trafficking in whole animals, and c) have potential as future photodiagnostic and/or phototherapeutic agents. In Phase I we will synthesize two classes of probes, one with FR (600-700 nm) excitation/emission and the other with MR (700-900 nm) excitation/emission. Each probe's cytotoxicity, membrane retention, signal:noise, and spectral overlap with commonly used antibody labels will be characterized using flow cytometry and confocal microscopy. Potential utility for macroscopic imaging will be assessed by determining image quality vs. depth in tissue "phantoms" containing varying admixtures of labeled and unlabeled cells. PROPOSED COMMERCIAL APPLICATIONS: Membrane labeling dyes emitting in the far red or near infrared have significant commercial potential in three markets: l) a research reagent market serving both basic and clinical investigators using flow cytometers; 2) a similar but growing market in optical imaging, especially macroscopic or whole body imaging; and 3) either photodiagnosis or phototherapy.